The major goal set forth in R01 GM079359 called for the development of molecular tools to elucidate the underlying mechanisms of diseases. Such tools would be engineered using aptamers, which are oligonucleic acids that bind to a specific target molecule. Accordingly, during the previous funding period, we successfully developed and applied a cell-based aptamer selection technique called cell-based Systematic Evolution of Ligands by Exponential enrichment (cell-SELEX) to generate multiple aptamers for the specific recognition of various types of diseased cells. Some of these aptamers were tested with clinical samples, and they showed promising results in differentiating normal from diseased samples. We also explored the use of aptamers in other biomedical applications, including bio analysis, molecular imaging, targeted drug development and biomarker discovery. Based on this work, we have published a total of 86 papers and filed 6 patents. Great strides were made during the previous funding period in developing aptamer probes that can recognize various types of diseased cells. However, we need to further systematically investigate the clinical, bioanalytical and therapeutic potential of these aptamer probes. This renewal request responds to that need. To accomplish this, we will (1) improve aptamer selection strategies, including new aptamer selections against intra- and extracellular proteins, (2) develop and optimize DNA aptamer-based technologies for bioanalytical and clinical applications such as molecular imaging (MI) and circulating tumor cell (CTC) analysis, and (3) conduct experiments aimed at perfecting i) aptamer-assisted biomarker discovery and ii) aptamer-conjugated radio isomers for combined chemotherapy and radiation therapy using aptamer-based drug delivery techniques recently discovered by our group. Our specific aims in this new research program are as follows: Aim 1. Develop and optimize DNA aptamers to recognize individual cells and proteins. Aim 2. Test the biomedical and biotechnological utility of aptamers in biomarker development, using membrane-bound proteins. Aim 3. Test and validate the clinical and bioanalytical utility of aptamers in circulating tumor cell detection and molecular imaging. Aim 4. Test the therapeutic utility of aptamers in targeted drug delivery, using an aptamer-radioactive isomer bioconjugate combined with chemotherapy for small cell lung cancer. Overall, the principle guiding this renewal application holds that a single technology (cell-SELEX) and a single modality (nucleic acid molecules, i.e., aptamers) can, when properly optimized, produce a mutually inclusive convergence of biomedical applications leading to one single end: the early detection, diagnosis and treatment of life-threatening diseases, in particular lung cancer. To carry out the aims in thi research program, we have already established a set of existing aptamers poised for optimization (see Table 1); however, over the course of the tenure of this work, conditions will call for the development of new aptamers for equally new applications, especially those required for the early detection of lung cancer subpopulations, as described below, requiring the extra and innovative step of pyrosequencing or ion semiconductor sequencing. To successfully conduct the necessary experiments, we have assembled a group of accomplished scientists and clinicians who have previously collaborated on many similar studies. Our project is innovative and rationally designed, our research goal is significant and important, our research and development in the last funding cycle are successful, and our preliminary results for future studies in this renewal are strong.